An in vitro model to evaluate the properties of matrices produced by fibroblasts from osteogenesis imperfecta and Ehlers-Danlos Syndrome patients.

ABSTRACT

AIM OF THE STUDY Osteogenesis imperfecta and Ehlers Danlos syndrome are hereditary disorders caused primarily by defective collagen regulation. Osteogenesis imperfecta patients were divided to haploinsufficient and dominant negative depending on the effect of COL1A1 and COL1A2 mutations whereas Ehlers Danlos syndrome patients had a mutation in PLOD1. Although collagen abnormalities have been extensively studied in monolayer cultures, there are no reports about 3D in vitro models which may reflect more accurately the dynamic cell environment. This is the first study presenting the structural and mechanical characterization of a 3D cell-secreted model using primary patient fibroblasts. MATERIALS AND METHODS: Fibroblasts from patients with osteogenesis imperfecta and Ehlers Danlos syndrome were cultured with ascorbic acid for 5 weeks. The effect of mutations on cytosolic and secreted collagen was tested by electrophoresis following incubation with radiolabeled 14C proline. Extracellular matrix was studied in terms of collagen fiber orientation, stiffness, as well as glycosaminoglycan and collagen content. RESULTS AND CONCLUSIONS: Osteogenesis imperfecta patients with haploinsufficient mutations had higher percentage of anisotropic collagen fibers alignment compared to other patient groups; all patients had a lower percentage of anisotropic samples compared to healthy controls. This correlated with higher average stiffness in the control group. Glycosaminoglycan content was lower in the control and haploinsufficient groups. In cells with PLOD1 mutations, there were no differences in PLOD2 expression. This proof of concept study was able to show differences in collagen fiber orientation between different patient groups which can potentially pave the way towards the development of 3D models aiming at improved investigation of disease mechanisms.